Artemisinin resistant kelch13 R622I and RDT negativity approaching predominance in northern Ethiopia and emerging C580Y of African origin threaten falciparum malaria control.

The rise of antimalarial drug-resistant Plasmodium falciparum poses a major threat to malaria treatment, control, and elimination efforts. Mutations in the kelch13 (k13) gene confer artemisinin partial resistance (ART-R), compromising the efficacy of combination therapies. In the Horn of Africa, the validated mutation k13 R622I has rapidly emerged in parallel with other mutations elsewhere in Eastern Africa. To monitor and inform control efforts, we conducted in-depth sampling where R622I was first detected, using molecular inversion probe (MIP) targeted sequencing of key resistance mutations and informative SNPs across the genome. Samples were collected for a year within two districts, Gondar Zuria and Tach Armachiho, representing different ecological zones in northwest Ethiopia. Among 903 people with P. falciparum malaria, we observed a markedly higher prevalence of R622I (44.3%) compared to earlier reports, with significantly higher prevalence in Gondar Zuria (52%) than in Tach Armachiho (40%; p<0.001). Alarmingly, the prevalence of histidine rich protein 2 (HRP2) based rapid diagnostic test (RDT) negativity was higher in R622I mutants compared to wild types (48.3% vs 30.7%; p<0.001). Furthermore, 98.2% of R622I samples carried the multidrug resistance protein 1 NFD haplotype associated with reduced susceptibility to lumefantrine. We also detected the k13 C580Y mutation in two patients (0.4%) from Gondar Zuria, the first report in the Horn of Africa. Identity-by-descent (IBD) analysis showed C580Y mutant parasites were likely clonal (IBD=1). Whole-genome sequencing confirmed their clonal nature and revealed flanking haplotypes distinct from those seen in Southeast Asia where the mutation was first observed, suggesting a local, de novo emergence. These findings highlight increasing prevalence and types of ART-R mutations and the concerning association now with RDT negativity that will further challenge malaria control and elimination efforts.